Prevention and Treatment of Oxidative Stress Disorders by Glutathione and Phase II Detoxification Enzymes

ABSTRACT

The present invention generally relates to the field of treating oxidative stress disorders by administering a pharmaceutically effective amount of a compound that elevates the intracellular levels of glutathone or intracelluar levels of at least one Phase II detoxification enzyme in animal tissue. The present invention also relates to the field of protecting a subject from oxidative stress disorders by administering a pharmaceutically effective amount of a compound that elevates the intracellular levels of glutathone or intracelluar levels of at least one Phase II detoxification enzyme in the subject. The present invention also relates to a pharmaceutical composition useful for the treatment of oxidative stress disorders.

The present application claims priority to U.S. provisional applicationSer. No. 60/340,273, filed on Dec. 18, 2001, which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention generally relates to the field of treatingoxidative stress by administering a pharmaceutically effective amount ofa compound that elevates intracellular levels of glutathione orintracellular levels of at least one Phase II detoxification enzyme inanimal cells. The present invention also relates to the field ofprotecting a subject from oxidative stress by administering apharmaceutically effective amount of a compound that elevatesintracellular levels of glutathione or intracellular levels of at leastone Phase II detoxification enzyme in animal cells. The presentinvention also relates to a pharmaceutical composition useful for thetreatment of an oxidative stress disorder.

BACKGROUND OF THE INVENTION

The toxicity of oxygen and more specifically its partial reductionproducts known as reactive oxygen species (ROS) is commonly designatedas oxidative stress. It arises from an imbalance of cellular pro-oxidantand antioxidant processes. Oxidative stress has been implicated in avariety of pathological and chronic degenerative processes including thedevelopment of cancer, atherosclerosis, inflammation, aging,neurodegenerative disorders, cataracts, retinal degeneration, drugaction and toxicity, reperfusion injury after tissue ischemia, anddefense against infection. See, for instance, Gao X, Dinkova-Kostova AT, Talalay P. (2001) Proc Natl Acad Sci USA., 98(26):15221-6, which isincorporated herein by reference. The publications listed at page 15226of Gao et al., 2001, supra, also are incorporated herein by reference.Mammalian cells contribute to their own oxidative stress by generatingROS as part of normal aerobic metabolism, and have developed elaborateand overlapping mechanisms for combating these hazards (Halliwell, B. &Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine.Oxford University Press, New York, pp. 1-36). Nevertheless, protectivemechanisms are not completely effective especially during increasedoxidative stress. The desirability of developing methods for augmentingthese defenses is reflected in the widespread human consumption andperceived health benefits of plant-based antioxidants such as ascorbicacid, tocopherols, carotenoids, and polyphenols (Pokorny, J.,Yanishlieva, M. & Gordon, M. (2001) Antioxidants in food: practicalapplications. Woodhead Publishing, Ltd., Cambridge, U.K). These directantioxidants neutralize free radicals and other chemical oxidants butare consumed in these reactions. Additional compounds are needed toprotect subjects from oxidative stress disorders as well as fortreatment of subjects suffering from these same disorders.

SUMMARY OF THE INVENTION

The invention relates to a method of treating a subject in need oftreatment of an oxidative stress disorder, which comprises administeringto the subject a pharmaceutically effective amount of a compound thatelevates glutathione, or at least one Phase II detoxification enzyme, inthe diseased tissue of the subject. The compound may be anisothiocyanate such as sulforaphane, or a glucosinolate. The oxidativestress disorder may be retinal degeneration, Alzheimer's Disease oraging. The Phase II detoxification enzyme may beUDP-glucuronosyltransferases, sulfotransferases,phenol-O-methyltransferase, catechol-O-methyltransferase, histamineN-methyltransferase, nicotinamide N-methyltransferase, thiopurinemethyltransferase, thiol methyltransferase, N-acetyltransferases,O-acetyltransferases, acyl-CoA synthetases, acyl-CoA:amino acidN-acyltransferases, aminoacyl-tRNA synthetases, glutathione synthetases,gamma glutamylcysteine synthetases, glutathione S-transferases, quinonereductases, heme oxygenases, rhodaneses, glutathione reductase,glutathione peroxidase, catalase and superoxide dismutase. The inventionrelates to a method of protecting a subject from oxidative stressdisorder, which comprises administering to the subject apharmaceutically effective amount of a compound that elevatesglutathione, or at least one Phase II detoxification enzyme, in thecells of the subject. The compound may be an isothiocyanate such assulforaphane, or a glucosinolate. The oxidative stress disorder may beretinal degeneration, Alzheimer's Disease or aging. The Phase IIdetoxification enzyme may be UDP-glucuronosyltransferases,sulfotransferases, phenol-O-methyltransferase,catechol-O-methyltransferase, histamine N-methyltransferase,nicotinamide N-methyltransferase, thiopurine methyltransferase, thiolmethyltransferase, N-acetyltransferases, O-acetyltransferases, acyl-CoAsynthetases, acyl-CoA:amino acid N-acyltransferases, aminoacyl-tRNAsynthetases, glutathione synthetases, gamma glutamylcysteinesynthetases, glutathione S-transferases, quinone reductases, hemeoxygenases, rhodaneses, glutathione reductase, glutathione peroxidase,catalase and superoxide dismutase.

The invention relates to a method of protecting a subject from oculardegeneration, which comprises administering to the subject apharmaceutically effective amount of a compound that elevatesintracellular levels of glutathione or intracellular levels of at leastone Phase II detoxification enzyme in diseased tissue of said subject.The compound may be an isothiocyanate such as sulforaphane, or aglucosinolate. The oxidative stress disorder may be retinaldegeneration, Alzheimer's Disease or aging. The Phase II detoxificationenzyme may be UDP-glucuronosyltransferases, sulfotransferases,phenol-O-methyltransferase, catechol-O-methyltransferase, histamineN-methyltransferase, nicotinamide N-methyltransferase, thiopurinemethyltransferase, thiol methyltransferase, N-acetyltransferases,O-acetyltransferases, acyl-CoA synthetases, acyl-CoA:amino acidN-acyltransferases, aminoacyl-tRNA synthetases, glutathione synthetases,gamma glutamylcysteine synthetases, glutathione S-transferases, quinonereductases, heme oxygenases, rhodaneses, glutathione reductase,glutathione peroxidase, catalase and superoxide dismutase.

Similarly, the present invention also relates to a method of protectinga subject from photooxidation, comprising administering to a subject apharmaceutically effective amount of a compound that elevatesintracellular levels of glutathione or intracellular levels of at leastone Phase II detoxification enzyme in a tissue of said subject. Thesubject's tissue may be skin, or an ocular organ, such as the eye.

The invention also relates to a composition for use in the treatment ofa oxidative stress disorder, which comprises a pharmaceutical excipientand a pharmaceutically effective amount of an agent that increasesintracellular levels of glutathione or at least one Phase IIdetoxification enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Protection of adult human retinal pigment epithelial (ARPE-19)cells against the toxicity of menadione (0-250 μM) by induction of Phase2 enzymes by sulforaphane (0-5 μM). Upper, Fractional killing of cells(f_(a)) as a function of menadione concentration at a series ofsulforaphane concentrations. Center, Analysis of the data by the medianeffect plot. The median effect concentrations (D_(m)) at the abovesulforaphane concentrations is shown. For sulforaphane concentrations5.00, 2.50, 1.25 and 0.63 μM, the D_(m) value is 134.2, 122.9, 109.9 and98.6 μM, respectively. Lower, Photograph of a typical 96-well microtiterplate showing the protective effect of sulforaphane against thecytotoxicity of menadione for human ARPE-19 cells. The intensity ofpurple color is the reduced MIT formazan for a measure of cellviability.

FIG. 2. Comparison of the effects of treatment of human ARPE-19 cellswith a series of concentrations of sulforaphane (0-5 μM) for 24 h on thetoxicity of exposure for 2 h to menadione. Left, cytotoxicity expressedas the median effect concentration (D_(m)). Center, glutathioneconcentrations expressed as nanomol/mg of cytosolic protein. Right,quinone reductase specific activity, expressed as nanomol/min/mg ofcytosolic protein. The multivariate regression correlations betweensulforaphane concentrations and the other three variables all had pvalues of <0.01.

FIG. 3. Prolonged protection of ARPE-19 cells against menadione toxicityby sulforaphane (SF) expressed as median effect concentrations (D_(m),μM). The menadione toxicity was determined immediately after induction(time=0), and 24, 48, 96 and 120 h later. Note that protection continuedto rise for 24-48 h, and then declined during the next 48 h.

FIG. 4. Persistent induction of quinone reductase (QR), glucose6-phosphate dehydrogenase (G6PD), glutathione reductase (GR)(nanomol/min/mg of cytosolic protein) and elevation of GSH levels(expressed as nanomol/mg of cytosolic protein) in human ARPE-19 cellsafter exposure to sulforaphane for 24 h[0,0 (▴), 0.625 (□) and 2.5 μM()].

FIG. 5. Protection of human ARPE-19 cells against the toxicity ofmenadione (62, 125, 200 μM for 2 h), tert-butyl hydroperoxide (0.5,0.75, 1 mM for 16 h), 4-hydroxynonenal (6.25, 12.5, 25 μM for 4 h), andperoxynitrite (1, 2, 4 mM for 2 h) as a function of prior exposure for24 h to 0-2.5 μM sulforaphane. The bar graphs show that cell viabilityis a function of both the concentrations of the oxidant and of thesulforaphane inducer. The front, center and rear series of bars refer tothe highest, middle and lowest concentration of oxidants, respectively.

FIG. 6. Protection of human keratinocytes (HaCaT) against oxidativecytotoxicity of tert-butyl hydroperoxide (0.313, 0.625, 1 mM for 8 h)(left), and mouse leukemia (L1210) cells against oxidative cytotoxicityof menadione (15.6, 31.3, 62.5 μM for 2 h) (right) by treatment withsulforaphane (0-2.5 μM for 24 h). The D_(m) and m values obtained fromthe median effect plots are shown in Table 1. The front, center and rearseries of bars refer to the highest, middle and lowest concentration ofoxidants, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method of treating a subject in needof treatment of an oxidative stress disorder, the method comprisingadministering to the subject a pharmaceutically effective amount of acompound that elevates levels intracellular glutathione or intracellularlevels of at least one Phase II detoxification enzyme in diseased tissueof said subject.

Many human chronic diseases are related to oxidative stress, but in someanatomical locations this oxidative stress is aggravated by light orirradiation. One common “anatomical location” is the skin, whereultraviolet light is a causative agent for skin cancer. Another exampleis constant damage in the retina of the eye by the retinal moleculesinvolved in the visual cycle. These polyunsaturated substances can actas oxidants (i.e., “photooxidants”) and their ability to producereactive oxygen species is markedly enhanced by UV light of theappropriated wavelength. We therefore studied whether the cytotoxiceffect of all-trans-retinal in combination with UV light could beabrogated by induction of phase 2 enzymes by sulforaphane. Example 6,described below, illustrates this protective function. In this respect,the present invention contemplates the abrogation of any cytotoxiceffect induced by retinal metabolic products, derivatives, or variantsthereof, such as “cis-retinal” (e.g., the 11-cis-retinal isoform), incombination with light, especially UV light, as prescribed by thepresent invention.

As used herein, the term subject can be used to mean an animal,preferably a mammal, including a human or non-human. The term patient isused to indicate a subject in need of treatment of an oxidative stressdisorder. The terms “disease,” “condition” and “disorder” may all beused interchangeably.

The current invention can be useful in treating a subject in need oftreatment of an oxidative stress disorder where there may be aberrantlevels of glutathione or any Phase II enzyme present in the diseasedcells or tissue. These abnormal levels may be either causal orsymptomatic of the oxidative stress disorder. The phrase “oxidativestress disorder,” as used in the current context, arises from animbalance of cellular pro-oxidant and antioxidant processes resulting incell death. Oxidative stress has been implicated in a variety ofpathological and chronic degenerative processes including thedevelopment of cancer, atherosclerosis, inflammation, aging,neurodegenerative disorders, cataracts, retinal degeneration, drugaction and toxicity, reperfusion injury after tissue ischemia, anddefense against infection.

The treatment envisioned by the current invention can be used forpatients with a pre-existing oxidative stress disorder, or for patientspre-disposed to an oxidative stress disorder. Additionally, the methodof the current invention can be used to correct cellular orphysiological abnormalities involved with an oxidative stress disorderin patients.

As used herein, the term agent or compound is intended to mean anychemical that elevates intracellular levels of glutathione or Phase IIenzymes.

As used herein, “a pharmaceutically effective amount” is intended usedto mean an amount effective to elicit a cellular response that isclinically significant, without excessive levels of side effects.

The present invention relates to methods of elevating intracellularglutathione (GSH) as a way to prevent or treat oxidative stressdisorders. GSH is a tripeptide composed of glycine, cysteine andglutamate, with the glutamate being linked to cysteine via thegamma-carboxyl group (as opposed to an alpha-carboxyl linkage thatnormally occurs is in a peptide). GSH is a detoxifying peptide that thebody conjugates to xenobiotics (foreign chemical or compound) to renderthe xenobiotic more hydrophilic, thus promoting their excretion from thebody. The synthesis of GSH involves a two-step reaction, with the firstbeing catalyzed by gamma-glutamylcysteine synthetase. Glutathionesynthetase catalyzes the second reaction. In turn, the conjugation ofGSH to the xenobiotic is catalyzed by glutathione S-transferase (alsoreferred to as GSH transferase), which can be a dimer of identical(homodimer) or different (heterodimer) subunits, although someheterodimers do exist. To date, at least four classes of GSHtransferases have been identified, with each class having two or moretypes of subunits therein. Most GSH transferases are cytosolic (orsoluble), meaning they are found in a cell's cytosol, although at leasttwo microsomal GSH transferases exist. Based on such factors as aminoacid similarity and biological activity, one of ordinary skill in theart will be able to appreciate and recognize the many types of GSHtransferases that exist, as well as any that may not yet be identified.Thus, as used herein, the phrases “increase in intracellular GSH” or“elevation in intracellular GSH” is intended to mean the GSH tripeptideitself, as well as the enzymes responsible for its synthesis andconjugation to xenobiotics.

The present invention also relates to a method increasing theintracellular levels of at least one Phase II detoxification enzyme. Thephrases “Phase II detoxification enzyme” and “Phase II enzyme” are usedinterchangeably herein. As used herein, a Phase II enzyme is any enzymethat is involved in any of the Phase II reactions which are responsiblefor the biotransformation of xenobiotics and/or prevention of oxidativestress. Generally speaking, a Phase II reaction will generally involvethe conjugation of a moiety to the xenobiotic that will increase thehydrophilicity of the xenobiotic. This conjugated xenobiotic, which isnow more hydrophilic, is more readily excreted from the body, than theunconjugated xenobiotic. There are six types of Phase II conjugationreactions, including glucuronidation, sulfation, methylation,acetylation, amino acid conjugation and glutathione conjugation. Thereaction catalyzed by the enzyme rhodanese (the transfer of a sulfur ionto cyanide to form thiocyanate) will also be considered a Phase IIreaction herein.

Briefly, glucuronidation is a major pathway of xenobiotic transformationand involves the conjugation of glucuronide to the xenobiotic. Thereaction is catalyzed by UDP-glucuronosyltransferase (UDPGT), of whichmultiple forms exist. These multiple forms are encoded by severaldifferent genes. One of ordinary skill in the art will be able torecognize and appreciate the different forms of UDPGT, and the reactionsthey catalyze.

Sulfation is a pathway of xenobiotic transformation that involves theconjugation of sulfate to the xenobiotic. The reaction is catalyzed bysulfotransferases, of which more than a dozen forms have beenidentified. One of ordinary skill in the art will be able to recognizeand appreciate the different forms of sulfotransferases, and thereactions they catalyze.

Methylation is a pathway of xenobiotic transformation that involves theconjugation of a methyl group to the xenobiotic. There are threedifferent methylation reactions, based on the type of atom on thexenobiotic that is methylated. The three methylation reactions can occuron, sulfur (S), oxygen (O) and nitrogen (N), each of which is catalyzedby a different sets of enzymes. O-methylation is catalyzed byphenol-O-methyltransferase (POMT) or catechol-O-methyltransferase(COMT). It is believed that COMT is encoded by a single gene, with atleast two different allelic variants. N-methylation is catalyzed byhistamine-N-methyltransferase or nicotinamide-N-methyltransferase.S-methylation is catalyzed by at least two enzymes, including thiopurinemethyltransferase (TPMT) and thiol methyltransferase (TMT). One ofordinary skill in the art will be able to recognize and appreciate thedifferent forms of methlytransferases, and the reactions they catalyze.

Acetylation is a pathway of xenobiotic transformation that involves theconjugation of an acetyl group to the xenobiotic. There are twodifferent acetylation reactions, based on the type of atom (O and N) onthe xenobiotic that is acetylated. The two acetylation reactions may ormay not be catalyzed by the same set of enzymes. N-acetylation iscatalyzed by N-acetyltransferase (NAT), of which two forms exist inhumans. These forms are encoded by two different genes. O-acetylation iscatalyzed by O-acetyltransferase, but may also be catalyzed by NAT. Oneof ordinary skill in the art will be able to recognize and appreciatethe different forms of acetyltransferases, and the reactions theycatalyze.

Amino acid conjugation is a pathway of xenobiotic transformation thatinvolves the conjugation of an amino acid to the xenobiotic. There aretwo principle pathways where amino acids are conjugated to xenobiotics.The first reaction involves xenobiotics containing a carboxylic acid.This reaction takes place by acyl-CoA synthetase catalyzing theconjugation of CoA to the xenobiotic to form a thioester. The thioesteris subsequently conjugated to the amino acid via the acyl-CoA: aminoacid N-transferase enzyme. The second principle pathway wherexenobiotics are conjugated to amino acids involves xenobioticscontaining an aromatic hydroxylamine. This reaction involves theactivation of an amino acid with the aminoacyl-tRNA-sythetase. Theactivated amino acid subsequently reacts with the aromatic hydroxylamineon the xenobiotic to form a reactive N-ester. One of ordinary skill inthe art will be able to recognize and appreciate the different types ofenzymes responsible for the conjugation of amino acids to xenobiotics,and the reactions they catalyze.

Quinone reductase (QR) is considered a Phase II enzyme because it hasprotective functions (Prochaska, et al., Oxidative Stress: Oxidants andAntioxidants, 195-211 (1991)), is induced coordinately with other phaseII enzymes, and is regulated by enhancer elements similar to those thatcontrol glutathione transferase (Favreau, et al., J. Biol. Chem.266:4556-4561 (1991)).

Heme oxygenase (HO), also considered a Phase H enzyme, catalyzes theconversion of heme to biliverdin, which is subsequently reduced tobilirubin. Thus the HO enzyme is responsible for the production ofbilirubin, which itself is a powerful antioxidant. Additionally, the HOenzyme is induced by many of the same compounds that induce other PhaseII enzymes.

Examples of additional Phase II enzymes include, but are not limited to,such enzymes as glutathione reductase, glutathione peroxidase, catalaseand superoxide dismutase.

The present invention relates to increasing the levels of at least onePhase II enzyme in diseased tissue. It is possible that the compoundscontemplated in this invention may be responsible for the increasedlevels of only one of the Phase II enzymes, or more than one Phase IIenzyme. As used herein, the phrase “increase in levels of Phase IIenzymes” is used to mean an increase in the quantity of Phase II enzymespresent in the cell, compared to control (non-stimulated) levels. Thephrase is also used to mean an increase in the activity or specificityof the enzymes present in the cell.

As used herein, the term diseased tissue may be used to mean individualcells, as cultured in vitro, or excised tissue in whole or in part.Diseased tissue may also be used to mean tissue in the subject that isundergoing the degenerative process, or tissue within the same organthat may not yet be affected by the degenerative process. The normaltissue may or may not be adjacent to the degenerative tissue.

As a preferred embodiment, the compounds used in the methods of thecurrent invention that elevate glutathione or any Phase II enzyme, areselected from the group consisting of an isothiocyanate and aglucosinolate.

Isothiocyanates are compounds containing the thiocyanate (SCN) moietyand are easily identifiable by one of ordinary skill in the art. Anexample of an isothiocyanate includes, but is not limited tosulforaphane or its analogs. The description and preparation ofisothiocyanate analogs is described in U.S. Reissue Pat. 36,784, and ishereby incorporated by reference in its entirety. In a preferredembodiment, the sulforaphane analogs used in the present inventioninclude 6-isothiocyanato-2-hexanone,exo-2-acetyl-6-isothiocyanatonorbornane,exo-2-isothiocyanato-6-methylsulfonylnorbornane,6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane,exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane,exo-2-acetyl-5-isothiocyanatonorbornane,1-isothiocyanato-5-methylsulfonylpentane,cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanate andtrans-3-(methylsulfonyl)cyclohexylmethylisothiocyanate.

Glucosinolates, which are well-known in the art, are precursors toisothiocyanates. Glucosinolates are easily recognizable and appreciatedby one of ordinary skill in the art and are reviewed in Fahey et al.Phytochemistry, 56:5-51 (2001), the entire contents of which are herebyincorporated by reference.

Other compounds contemplated by the present invention include compoundsthat are known to induce (increase) levels of Phase II enzymes.Preferably, these compounds include resveratrol, oltipraz,dimethylfumarate, 2(3)-tert-butyl-4-hydroxyanisole,3,5-di-tert-butyl-4-hydroxytoluene and an analog thereof.

As used herein, the phrase “increased or decreased expression” is usedto mean an increase or decrease in the transcription rates of the genesresponsible for coding the enzymes, resulting in an increase or decreasein the levels of mRNA for each gene, respectively. The phrase is alsoused to mean an increase or decrease in the levels of the protein orenzyme in the cell, independent of transcription rates. For example, anincrease in degradation rate of an mRNA encoding the protein inquestion, without a change in the transcription rate, may result in adecrease in the levels of protein in the cell.

The current invention also provides for a composition for use in thetreatment of an oxidative stress disorder, comprising a pharmaceuticalexcipient and a pharmaceutically effective amount of an agent thatincreases intracellular levels of glutathione or at least one Phase IIdetoxification enzyme.

In a preferred embodiment, the composition of the current inventioncomprises an agent selected from the group consisting of anisothiocyanate and a glucosinolate. In a more preferred embodiment, thecomposition of the current invention comprises sulforaphane or asulforaphane analog.

In another preferred embodiment, the composition of the currentinvention comprises an agent selected from the group consisting of6-isothiocyanato-2-hexanone, exo-2-acetyl-6-isothiocyanatonorbornane,exo-2-isothiocyanato-6-methylsulfonylnorbornane,6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane,exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane,exo-2-acetyl-5-isothiocyanatonorbornane,1-isothiocyanato-5-methylsulfonylpentane,cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanate andtrans-3-(methylsulfonyl)cyclohexylmethylisothiocyanate.

In still another preferred embodiment, the composition of the currentinvention comprises an agent selected from the group consisting ofresveratrol, oltipraz, dimethylfumarate,2(3)-tert-butyl-4-hydroxyanisole, 3,5-di-tert-butyl-4-hydroxytoluene andan analog thereof.

In another preferred embodiment, the composition of the currentinvention, which is used to treat degenerative diseases by increasingthe levels of glutathione or any Phase II enzyme, is used to increasethe enzymes selected from the group consisting ofUDP-glucuronosyltransferases, sulfotransferases,phenol-O-methyltransferase, catechol-O-methyltransferase, histamineN-methyltransferase, nicotinamide N-methyltransferase, thiopurinemethyltransferase, thiol methyltransferase, N-acetyltransferases,acyl-CoA synthetases, acyl-CoA:amino acid N-acyltransferases,aminoacyl-tRNA synthetases, glutathione synthetases, gammaglutamylcysteine synthetases, glutathione S-transferases, quinonereductases, heme oxygenases and rhodaneses.

Formulations and Methods of Administration

A pharmaceutical composition of the invention is provided comprising anagent of the invention useful for treatment of an oxidative stressdisorder and a pharmaceutically acceptable carrier or excipient. Such anagent may be artificially synthesized or may be obtained from anaturally occurring source. If the agent is obtained from a naturallyoccurring source it may not be necessary to combine such agent with apharmaceutically acceptable carrier or excipient. An exemplary naturallyoccurring source is Brassica oleracea seeds selected from the group ofvarieties consisting of acephala, alboglabra, botrytis, costata,gemmifera, gongylodes, italica, medullosa, palmifolia, ramosa, sabauda,sabellica, and selensia. An additional exemplary naturally occurringsource is cruciferous sprouts.

The invention further contemplates the use of prodrugs which areconverted in vivo to the therapeutic compounds of the invention(Silverman, R. B., “The Organic Chemistry of Drug Design and DrugAction,” Academic Press, Ch. 8 (1992)). Such prodrugs can be used toalter the biodistribution or the pharmacokinetics of the therapeuticcompound. For example, an anionic group, e.g., a sulfate or sulfonate,can be esterified, e.g, with a methyl group or a phenyl group, to yielda sulfate or sulfonate ester. When the sulfate or sulfonate ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, to reveal the anionic group. Such an ester can becyclic, e.g., a cyclic sulfate or sultone, or two or more anionicmoieties can be esterified through a linking group. An anionic group canbe esterified with moieties (e.g., acyloxymethyl esters) which arecleaved to reveal an intermediate compound which subsequently decomposesto yield the active compound. Furthermore, an anionic moiety can beesterified to a group which is actively transported in vivo, or which isselectively taken up by target organs. The ester can be selected toallow specific targeting of the therapeutic moieties to particularorgans, as described below for carrier moieties.

The pharmaceutical composition can be administered orally, nasally,parenterally, intrasystemically, intraperitoneally, topically (as bydrops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is intended, but not limited to, anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

A pharmaceutical composition of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

The compositions of the present invention can also contain adjuvantssuch as, but not limited to, preservatives, wetting agents, emulsifyingagents, and dispersing agents. Prevention of the action ofmicroorganisms can be ensured by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It can also be desirable to include isotonicagents such as sugars, sodium chloride, and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of the drugs, it isdesirable to slow the absorption from subcutaneous or intramuscularinjection. This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include, but are not limitedto, capsules, tablets, pills, powders, and granules. In such soliddosage forms, the active compounds are mixed with at least one itempharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, acetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and I) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hardfilled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, solutions, suspensions,syrups and elixirs. In addition to the active compounds, the liquiddosage forms can contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Administration to the eyes canbe any of the many methods well know to those skilled in the artincluding drops, foams, polymeric compositions, gels, implantabletime-release compostions, oral dose forms, injectable dose forms, phasetransition forms, ointments, creams, solid implants, among others.

Compositions for topical administration, including those for inhalation,can be prepared as a dry powder which can be pressurized ornon-pressurized. In nonpressurized powder compositions, the activeingredients in finely divided form can be used in admixture with alarger-sized pharmaceutically acceptable inert carrier comprisingparticles having a size, for example, of up to 100 μm in diameter.Suitable inert carriers include sugars such as lactose. Desirably, atleast 95% by weight of the particles of the active ingredient have aneffective particle size in the range of 0.01 to 10 μm.

Alternatively, the composition can be pressurized and contain acompressed gas, such as nitrogen or a liquefied gas propellant. Theliquefied propellant medium and indeed the total composition ispreferably such that the active ingredients do not dissolve therein toany substantial extent. The pressurized composition can also contain asurface active agent. The surface active agent can be a liquid or solidnon-ionic surface active agent or can be a solid anionic surface activeagent. It is preferred to use the solid anionic surface active agent inthe form of a sodium salt.

Dosaging

One of ordinary skill will appreciate that effective amounts of theagents of the invention can be determined empirically and can beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt, ester or prodrug form. The agents can be administeredto a subject, in need of treatment of a neurological disorder, aspharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of the agents orcomposition of the present invention will be decided by the attendingphysician within the scope of sound medical judgement. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors: the type and degree of the cellularresponse to be achieved; activity of the specific agent or compositionemployed; the specific agents or composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of theagent; the duration of the treatment; drugs used in combination orcoincidental with the specific agent; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the agents at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosagesuntil the desired effect is achieved.

For example, satisfactory results are obtained by oral administration ofthe compounds at dosages on the order of from 0.05 to 10 mg/kg/day,preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day,most preferably 0.5 mg/kg/day administered once or, in divided doses, 2to 4 times per day. On administration parenterally, for example by i.v.drip or infusion, dosages on the order of from 0.01 to 5 mg/kg/day,preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0mg/kg/day can be used. Suitable daily dosages for patients are thus onthe order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., morepreferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v.,preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.

Dosaging can also be arranged in a patient specific manner to provide apredetermined concentration of the agents in the blood, as determined bytechniques accepted and routine in the art (HPLC is preferred). Thuspatient dosaging can be adjusted to achieve regular on-going bloodlevels, as measured by HPLC, on the order of from 50 to 1000 ng/ml,preferably 150 to 500 ng/ml.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein can be made without departing from thescope of the invention or any embodiment thereof.

The following Examples serves only to illustrate the invention, and arenot to be construed as in any way to limit the invention.

EXAMPLES

Retinal cells are especially sensitive to oxidative damage (Cai, J.,Nelson, K. C., Wu, M., Sternberg, P. & Jones, D. P. (2000) Prog. RetinalEye Res. 19, 205-221, 2000; Winkler, B. S., Boulton, M. E., Gottsch, J.D. & Sternberg, P. (1999) Molecular Vision 5, 32-42.). To mimic thetypes of oxidative stresses that occur physiologically, we selected thefollowing four oxidants: menadione, tert-butyl hydroperoxide,4-hydroxynonenal, and peroxynitrite. The mechanisms by which theseagents evoke oxidative damage, and how cells protect themselves againstsuch damage are quite different, as described below.

In the examples, ARPE-19 cells were treated with sulforaphane, anisothiocyanate isolated from broccoli on the basis of its Phase 2inducing activity and the most potent naturally occurring Phase 2 enzymeinducer identified to date (Fahey, J. W., Zhang, Y. & Talalay, P. (1997)Proc. Natl. Acad. Sci. USA 94, 10367-10372; Zhang, Y., Talalay, P., Cho,C. G. & Posner, G. H. (1992) Proc. Natl. Acad. Sci. USA 89, 2399-2403;and Zhang, Y., Kensler, T. W., Cho, C. C., Posner, G. H. & Talalay, P.(1994) Proc. Natl. Acad. Sci. USA 91, 3147-3150). Sulforaphanecoordinately induces a family of Phase 2 detoxication enzymes andrelated proteins, and raises GSH levels by inducing γ-glutamylcysteinesynthetase, the rate-limiting enzyme in GSH biosynthesis (Mulcahy, R.T., Wartman, M. A., Bailey, H. H. & Gipp, J. J. (1997) J. Biol. Chem.272, 7445-7454).

Cell viability measurements were analyzed by the median effect equationof Chou and Talalay, (1984) Adv. Enzyme Regul. 22, 27-55, in order toobtain the median effect concentration (D_(m)) based on all the datapoints of the cytotoxicity-concentration curves. The D_(m) value foreach oxidant was then compared to that for cells that had been treatedwith a range of concentrations of sulforaphane, thereby generatingquantitative measures of protection.

Sulforaphane cannot react directly with free radicals or ROS: its“antioxidant” function is secondary to its ability to induce Phase 2enzymes, and it is therefore an “indirect antioxidant.” The magnitude ofthe protective effects depends on concentrations of both oxidantstressors and inducers. Notably, unlike the effects of most directantioxidants, the indirect antioxidant status persists for several daysafter sulforaphane is no longer present.

Parallel measurements of Phase 2 enzymes and GSH levels were obtained oncell extracts that had been exposed to sulforaphane under identicalconditions to those used in protection experiments. When the degree ofprotection, quantified by increases in D_(m) values, was compared toelevations of these Phase 2 markers, remarkably close correlations wereobserved. Taken together, these results establish that protectionagainst oxidative stress is quantitatively related to the indirectantioxidant action of sulforaphane which results from elevations ofPhase 2 enzymes and GSH.

Materials And Methods

Chemicals. Tert-Butyl hydroperoxide, 3-morpholinosydnonimine (SIN-1),menadione sodium bisulfite (menadione), and3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT),all-trans-retinal was purchased from Sigma (St. Louis, Mo.).4-Hydroxynon-2-enal was obtained from Cayman Chemical Co. (Ann Arbor,Mich.), and synthetic sulforaphane[1-isothiocyanato-(4R,S)-(methylsulfinyl)butane] was from LKTLaboratories (St. Paul, Minn.).

Cell Culture.

Human adult retinal pigment epithelial cells (ARPE-19, TCC Catalog No.CRL-2302) were obtained from the American Type Culture Collection(Manassas, Va.). These cells have structural and functional propertiessimilar to the analogous retinal cells in vivo (Dunn, K. C.,Aotaki-Keen, A. E., Putkey, F. R. & Hjelmeland, L. M. (1996) Exp. EyeRes. 62, 155-159). They were cultured in a mixture of equal volumes ofDulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium plus 10%fetal bovine serum that was heated for 90 min at 55° C. with 1% (w/v)charcoal.

Human skin keratinocytes (HaCaT) were obtained from Dr. G. Tim Bowden,Arizona Cancer Center, Tucson, Ariz. and grown in Eagle's minimumessential medium (EMEM) plus 8% fetal bovine serum that had been treatedwith Chelex resin (Bio-Rad, Hercules, Calif.) to remove Ca²⁺ (Boukamp,P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A. &Fawning, N. B. (1988) J. Cell Biol. 106, 761-771). Mouse L1210 leukemiacells, a gift from Dr. Joseph G. Cory, East Carolina State University,Greenville, N.C., were grown in RPMI 1640 medium supplemented with 10%horse serum. All cultures were incubated in a humidified atmosphere of5% CO₂ at 37° C. Media and sera were obtained from Life Technologies(Rockville, Md.).

Induction of Phase 2 Response by Sulforaphane.

All experiments were performed in 96-well microliter plates. ARPE-19 andHaCaT cells were seeded at 10,000 cells per well and grown for 24 hbefore addition of sulforaphane, whereas L1210 cells (5,000 cells/well)were not incubated before sulforaphane treatment. Solutions ofsulforaphane (5 mM) in dimethyl sulfoxide were diluted with the cognateculture medium to provide final inducer concentrations of 0.16-5.0 μM.The final DMSO concentrations were 0.1% by volume.

Choice of Oxidants.

tert-Butyl hydroperoxide differs from lipid hydroperoxides in beingwater-soluble, but unlike hydrogen peroxide, it is not metabolized bythe peroxidative actions of catalase. It is principally inactivated bydirect and glutathione transferase-promoted reduction of GSH (Hurst, R.,Bao, Y., Jemth, P., Mannervik, B. & Williamson, G. (1998) Biochem J.332, 97-100). Menadione causes necrotic cell death by participating inoxidative cycling which generates superoxide and more reactive oxygenspecies, by depletion of sulfhydryl groups, and by accumulation of toxicintracellular levels of calcium (Smith, M. T., Evans, C. G., Thor, H. &Orrenius, S. (1985) In: Oxidative stress, (H Sies, ed.) Academic Press,London, pp. 91-113). The relative toxicological importance of theseprocesses probably depends on the tissue and local conditions. Animportant detoxification mechanism for menadione is the obligatorytwo-electron reduction to hydroquinones promoted by NAD(P)H: quinonereductase 1 (QR) (Dinkova-Kostova, A. T. & Talalay, P. (2000)FreeRadical Biol. Med. 29, 231-240). Mice in whom this gene has beendisrupted are much more sensitive to the toxicity of menadione(Radjendirane, V., Joseph, P., Lee, Y.-H., Kimura, S., Klein-Szanto, A.J. P., Gonzalez, F. J. & Jaiswal, A. K. (1998) J. Biol. Chem. 273,7382-7389).

4-Hydroxynonenal is a highly cytotoxic and genotoxic alkenal that arisesfrom peroxidation of polyunsaturated fatty acids such as arachidonicacid and its tissue abundance is widely used as an index of lipidperoxidation (Prior, W. A. & Porter, N. A. (1990) Free Radical Biol.Med. 8, 541-543; Esterbauer, H., Schauer, R. J. & Zollner, H. (1991)Free Radical Biol. Med. 11, 81-128). The principal pathway fordetoxification of 4-hydroxynonenal is conjugation with GSH byglutathione transferases leading to mercapturic acid formation (Alary,J., Bravais, E., Cravedi, J. P., Debrauwer, L., Rao, D. & Bories, G.(1995) Chem. Res. Toxicol. 8, 34-39; Hubatsch, I., Ridderstrom, M. &Mannervik, B. (1998) Biochem J. 330, 175-179).

Peroxynitrite is a much more powerful oxidant than either superoxide ornitric oxide and is formed in cells by the exceedingly rapid combinationof these molecules. Although nitric oxide can protect cells againstapoptosis, peroxynitrite is a much more toxic reagent and attacks manycellular components, reacting with thiols, iron-sulfur centers, and zincfingers, and it initiates lipid peroxidation. It also nitrates tyrosineby a reaction catalyzed by superoxide dismutase (Estévez, A. G., Spear,N., Pelluffo, H., Kamaid, A., Barbeito, L. & Beckman, J. S. (1999)Methods Enzymol. 301, 393-402). Peroxynitrite probably generatescellular oxidative stress by several mechanisms.

There is accumulating evidence that chronic exposure to sunlight plays arole in some degenerative diseases, for instance, age-related maculardegeneration (AMD). We have, therefore, carried out an experiment ofprotection of human RPE cells against photooxidative damage mediated byall-trans-retinal, which exists in human retina and is a criticalcomponent of the visual cycle.

Treatment With Oxidants. Tert-Butyl hydroperoxide (1 M) and4-hydroxynonenal (25 mM) were dissolved in DMSO and diluted 1000-foldwith serum-free medium before addition of serial dilutions to themicrotiter plate wells. The final concentrations of DMSO were thereforeless than 0.1% (by vol.). Menadione sodium bisulfite (0.5 M) and SIN-1(0.5 M) were dissolved and added in PBS. ARPE-19 cells were exposed tomenadione for 2 h and to tert-butyl hydroperoxide for 16 h, washed withPBS, and cell viability was determined by the MIT test. ARPE-19 cellswere exposed to peroxynitrite for 2 h and 4-hydroxynonenal for 4 h, andthe cells were then incubated in serum-free media for 22 and 20 h,respectively, washed with PBS, and cell viability was determined. Theadditional incubation periods were required for peroxynitrite and4-hydroxynonenal to evoke maximal cytotoxicity.

Cytotoxicity Measurements.

Cell viability was determined by spectroscopic measurement of thereduction of MTT (Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna,J. D. & Mitchell, J. B. (1987) Cancer Res. 47, 936-942). The culturemedia were discarded after the designated incubation periods, the cellswere washed three times with PBS by use of a microtiter plate washer(Ultrawash Plus, Dynex Technologies, Chantilly, Va.). Each well thenreceived 150 μl of an MTT solution (0.5 mg/ml) in serum-free medium. Theplates were incubated for 2 h at 37° C., the MTT solution was discarded,100 μl of DMSO were added to each well, and the plates were shaken at200 rpm on an orbital shaker for 5 min. The absorbances of the wellswere determined at 555 nm with a microtiter plate reader (Spectra MaxPlus, Molecular Devices, Sunnyvale, Calif.). The absorbance of reducedMIT was then compared at each inducer and oxidant concentration to thatof untreated control cells that received only the vehicle in whichsulforaphane (DMSO) and menadione (DMSO or PBS) were dissolved. In eachexperiment three identical 96-well plates were used and the means of theabsorbance values, the standard deviations of these means, and theircoefficients of variation were calculated. The coefficients of variationranged from 0.6% to 16.5%. The mean coefficients of variation weresimilar for treated and untreated cells and averaged 7.2±4.2%.

Quantitative Analysis of Cytotoxicity.

Dose-effect analyses were performed according to the Median EffectEquation, by use of a computer program (Chou, T. C. & Hayball, M. (1996)CalcuSyn for Windows 3.1 and 95: multiple dose effect analyzer andmanual for IBM-PC, Biosoft, Cambridge, U.K). The equation:f_(a)/f_(u)=[D/D_(m)]^(m), where f_(a) is the fraction of cells affectedby the oxidant, f_(u) is the fraction unaffected (i.e., 1-f_(a)), D isthe dose of oxidant required to produce the effect f_(a), D_(m) is theconcentration of oxidant required to produce a 50% effect, i.e., whenf_(a)=f_(u), and the slope m is a measure of the sigmoidicity of thedose-response curve, and is therefore a measure of cooperativity. Theresults are analyzed by plotting log(f_(a)/f_(u)) with respect to log Dof the oxidant. The computer program provides the slope (m) of thecurves, and the goodness of fit (r²) to linearity.

Preparation Of Cell Lysates.

Cells were lysed by addition of a digitonin solution (0.8 mg,/mldigitonin in 2 mM EDTA, pH 7.8), incubated at 37° C. for 20 min, gentlyshaken for 20 min at 25° C., and centrifuged at 1,500×g for 20 min at 4°C.

Glutathione Analysis.

Total glutathione (oxidized and reduced) was determined by reduction of5,5′-dithiobis-2-nitrobenzoic acid (DTNB) in a glutathionereductase-coupled assay in 96-well microliter plates (Ritchie, J. P.,Jr., Skowronski, L., Abraham, P. & Leutzinger, Y. (1996) Clin. Chem. 42,64-70). Briefly, 30 μl of lysates were mixed with 60 μl of cold 2.5%metaphosphoric acid, stored at 4° C. for 10 min, and centrifuged for 20min at 1,500×g at 4° C. In a new plate, 50 μl of the supernatantfraction of each sample were mixed with 50 μl of 1.26 mM DTNB, 50 μl of200 mM sodium phosphate, pH 7.5, 5 mM EDTA, and 50 μl of a solutioncontaining 3.1 units/ml of yeast glutathione reductase (Sigma, St.Louis, Mo.). After 5 min incubation at 25° C., 50 μl of 0.72 mM NADPHwere added to each well, and the initial reaction rates were determinedat 412 nm. Calibration curves for pure GSH were included in each assay.

Enzyme Assays.

All measurements were made in 96-well microtiter plates at 25° C., andreaction rates were monitored with a microtiter plate reader. The QRactivities of supernatant fractions were determined by proceduresdeveloped in our laboratory (Fahey, J. W., Zhang, Y. & Talalay, P.(1997) Proc. Natl. Acad. Sci. USA 94, 10367-10372; Prochaska, H. J.,Santamaria, A. B. & Talalay, P. (1992) Proc. Natl. Acad. Sci. USA 89,2394-2398.). Specific activities were obtained by relating the reactionrates to protein concentrations determined with the bicinchoninic acidreagent (Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,Gratner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson,B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, 76-85). Thedicumarol-inhibitable fraction of the total QR activity contributed morethan 90% to the overall observed rates in the ARPE-19, HaCaT, and L1210cells. Glutathione reductase activity was assayed by mixing 50 μl ofcell lysate with 25 μl of 1 mM NADPH, 25 μl of GSSG (20 mg/ml), and 150μl of 50 mM sodium phosphate, pH 7.5. Initial reaction rates wereobtained at 340 nm (Carlberg, I. & Mannervik, B. (1985) Methods Enzymol.113, 484-490). Glucose 6-phosphate dehydrogenase was assayed by mixing50 μl of cell lysate with 200 μl of assay buffer containing 2.0 mMglucose 6-phosphate, 20 mM MgCl₂, and 150 μM NADP. Initial reactionrates were determined at 340 nm (Kornberg, A. & Horecker, B. L. (1955)Methods Enzymol. 1, 323-327).

Example 1 Quantitative Measurements of Menadione Toxicity to HumanRetinal Pigment Epithelial Cells and Protection by Sulforaphane

A standardized, highly reproducible system for quantitativedetermination of oxidant toxicity and protection by sulforaphane wasdeveloped for ARPE-19 cells grown in 96-well microtiter plates. Theprotective effect of 24 h prior incubation with 0-5 μM concentrations ofsulforaphane on survival of ARPE-19 cells exposed for 2 h to 0-250 μMmenadione is illustrated in FIG. 1, which shows the S-shaped dependenceof cytotoxicity on increasing concentrations of menadione (plotted asthe fractional cell kill, or fraction affected=f_(a)). At the highestconcentration of menadione almost no cells survive, but prior treatmentwith sulforaphane protected a substantial fraction of cells againstoxidative death. Over the concentration ranges examined, cell survivaldecreases as the concentration of the oxidant menadione is increased,and increases as the concentration of sulforaphane is raised, as shownin the photograph (FIG. 1).

Analysis of the data by the median effect equation of Chou & Talalay,(1984) Adv. Enzyme Regul. 22, 27-55, provides: (a) a measure of thetoxicity of the oxidant under each set of experimental conditions,expressed as the median effect concentration (D_(m)); (b) compliance ofthe data with mass action principles that underlie the theoretical basisof the median effect equation i.e., the magnitudes of r² values of plotsof log [f_(a)/f_(u)] with respect to log D; and (c) the Hill typecoefficient (m), a measure of the sigmoidicity of the curves and henceof the cooperativity between the processes contributing to thebiological endpoint (cell death). The median effect plots of the abovedata (FIG. 1) are a family of parallel and linear graphs (average of r²for 5 plots=0.976±0.016) with average slopes (m) of 3.44±0.22 (Table 1,shown below). These high slopes suggest that the processes contributingto cytotoxicity of menadione are highly cooperative. Notably, the D_(m)values rise asymptotically from 72.2 μM menadione under basal conditionsto 134.2 μM for cells that had been treated with 5 μM sulforaphane for24 h. In two other experiments, carried out at intervals of many weeks,the control D_(m) values were 65.0 and 69.0 μM, respectively, and weretherefore in good agreement.

Table 1 also shows results from experiments in which ARPE-19 cells,HaCaT cells or L1210 cells were treated with the Menadione, tert-Butylhydroperoxide, 4-hydroxynonenal, or peroxynitrite. The viability of thecells was determined by the MTT reduction measurements under conditionsdescribed above. D_(m) values were obtained from a series of plots oflog(f_(a)/f_(u)) with respect to log oxidant concentration at eachconcentration of sulforaphane. The m values are the slopes of theseplots and r² the linear correlation coefficients.

TABLE 1 Analysis by Median Effect Equation of protection by sulforaphaneof human retinal pigment epithelial cells (ARPE-19), keratinocytes(HaCaT), and murine leukemia (L 1210) cells, against the toxicities ofmenadione, tert-butyl hydroperoxide, 4-hydroxynonenal and peroxynitriteOxidants Sulforaphane (μM) D_(m) (μM) m r² ARPE-19 cells Menadione 0.0072.2 3.35 0.952 0.63 98.6 3.69 0.979 1.25 110 3.49 0.983 2.50 123 3.580.994 5.00 134 3.12 0.972 tert-Butyl 0.00 95.8 2.52 0.923 hydroperoxide0.63 140 2.17 0.964 1.25 163 1.79 0.980 2.50 165 1.26 0.9534-hydroxynonenal 0.00 8.70 2.85 0.885 0.63 14.1 2.51 0.931 1.25 25.82.78 0.981 2.50 26.8 2.51 0.993 Peroxynitrite 0.00 1440 6.07 0.958 0.632780 6.30 0.984 1.25 2820 6.19 0.982 2.50 2890 6.62 0.977 HaCaT celltert-Butyl 0.00 63.5 0.899 0.955 hydroperoxide 0.63 113 0.894 0.965 1.25166 0.921 0.971 2.50 200 0.768 0.974 L1210 cell Menadione 0.00 12.20.725 0.967 0.16 19.6 0.864 0.986 0.31 26.5 1.00 0.987 0.63 36.2 1.170.977

Example 2 Correlation Between Protection of ARPE-19 Against MenadioneToxicity by Sulforaphane and Elevations of Glutathione Levels andQuinone Reductase Specific Activities

The specific activities of QR and concentrations of GSH were measured incytosols of ARPE-19 cells that had been treated with 0-5.0 μMsulforaphane for 24 h, under conditions identical to those used above todetermine the D_(m) values for menadione toxicity. As expected, bothindicators of Phase 2 induction rose with exposure to increasingconcentrations of sulforaphane (FIG. 2). The responses were linearlycorrelated with the sulforaphane concentration (r²=0.995 and 0.935,respectively). More importantly, a multivariate regression analysisshowed a highly correlation between sulforaphane concentrations and QRactivities, GSH levels and D_(m) values (p=0.0095, 0.0004, 0.0038,respectively). There is therefore a highly significant quantitativeassociation between the degree of protection afforded by sulforaphaneagainst menadione toxicity and the elevations of QR activities and GSHlevels, suggesting strongly that the changes in these variables arecausally related.

Example 3 Sulforaphane Provides Prolonged Antioxidant Protection AgainstMenadione Oxidant Stress

Since sulforaphane, like other isothiocyanates, does not normallyparticipate in oxidation/reduction reactions, its antioxidant mechanismmust be indirect, presumably through induction of Phase 2 proteins.Consequently, it seemed likely that the protective effects ofsulforaphane should be catalytic and persist for several days (inrelation to the half-lives of the cognate proteins) after removal of theinducer, unlike direct antioxidants (e.g., ascorbic acid, tocopherols)which are consumed stoichiometrically in radical quenching reactions.Therefore ARPE-19 cells were treated for 24 h with two concentrations ofsulforaphane (0.625 and 2.5 μM) and then incubated them for anadditional 96 h in medium without fetal bovine serum (in order tominimize the complications of cell growth and the difficulties ofdistinguishing the effects of cell mass increases on specificbiochemical indices). Triplicate sets of identical plates were evaluatedfor menadione toxicity (2-h exposure) immediately after sulforaphaneexposure and at 24-h intervals thereafter. The median effectconcentration (D_(m)) for menadione of control cells was 66.8 μM and theD_(m) values for cells treated with 0.625 and 2.5 μM sulforaphane were69.2 and 94.5 μM, respectively. Control cell resistance remainedunchanged for 48 h, whereas the resistance to menadione toxicity of thecells treated with sulforaphane continued to increase during thisperiod, and then declined over the subsequent 48 h, finally approachingcontrol cell levels (FIG. 3).

These experiments establish that the protection evoked by sulforaphaneat the end of the 24-h induction treatment is maintained or exceeded forat least 3 days in culture (FIG. 3). The specific activities of QR,glucose 6-phosphate dehydrogenase, and glutathione reductase in thecytosols of cells treated in an identical manner also continued to risefor 48 h after removal of sulforaphane from the medium and then remainedhigh (glucose 6-phosphate dehydrogenase and glutathione reductase) ordeclined modestly (QR) over the ensuing 48-72 h (FIG. 4). The GSH levelsafter 24 h treatment with 2.5 μM sulforaphane were increased about 50%,remained at this level for another 24 h, and then declined to controlcell levels in the ensuing 96 h. Notably, in ARPE-19 cells that havebeen exposed to sulforaphane for 24 h, and are then maintained inserum-free culture media for several days, the protective status remainssubstantially elevated, in parallel with higher levels of GSH andelevated Phase 2 enzyme markers.

Example 4 Protection of ARPE-19 Cells by Sulforaphane Against theOxidative Stress of Tert-Butyl Hydroperoxide, Peroxynitrite, and4-Hydroxynonenal by Sulforaphane

Treatment of ARPE-19 cells with a range of concentrations ofsulforaphane (0, 0.625, 1.25, and 2.5 mM) for 24 h, also providedprotection against other oxidants with mechanisms of action thatdiffered from that of menadione. Thus the cytotoxicities of tert-butylhydroperoxide (0.5-1.0 mM for 16 h), peroxynitrite (generated fromSIN-1, 0.25-4.0 mM for 2 h), and 4-hydroxynonenal (1.56-25 μM for 4 h)were also significantly ameliorated by treatment with sulforaphane. Thisprotection, like that against menadione, depended on concentration ofboth the oxidants and sulforaphane (FIG. 5 and Table 1).

More detailed examination of the protective effects by the median effectequation reveals: (a) the slopes m for the cytotoxicities of theseoxidants are quite different (means of 1.93, 2.66, and 6.29 fortert-butyl hydroperoxide, 4-hydroxynonenal, and peroxynitrite,respectively), and different from the m value (3.45) for menadione; and(b) the degree of protection provided by comparable concentrations ofsulforaphane against different antioxidants ranged from 2- to 3-fold.

Example 5

Protection of Human Keratinocytes (HaCaT) and Murine Leukemia (L1210)Cells Against Oxidative Stress

To examine the generality of protection by Phase 2 induction an analysisof the effects of 24 h treatment with sulforaphane on the toxicity tohuman keratinocytes (HaCaT) and mouse leukemia (L1210) cells oftert-butyl hydroperoxide and menadione, respectively (FIG. 6) wasconducted. Interestingly, the slopes of the median effect plots for bothoxidants in these cell lines are in the 0.8-1.2 range, indicating lackof significant cooperativity among the processes contributing to celldeath in these cell lines. This is quite different from the effects ofthe same oxidants on ARPE-19 cells (Table 1). It appears therefore thatthe cooperativity between lethal processes depends on the cell line.Nevertheless, the substantial protection observed in the untransformedhuman keratinocyte cell line and in the highly neoplastic murineleukemia cell line indicates that the protection provided bysulforaphane is a more general phenomenon, not restricted to retinalepithelial pigment cells.

Example 6 Protection of Human ARPE Cells Against the Photo-OxidativeAttack Induced by all-Trans-Retinal and Light Exposure at 365 Nm

To examine the protective effect of sulforaphane against photooxidativedamage mediated by all-trans-retinal and light exposure, the cells weretreated with 0-100 μM of all-trans-retinal for 2 hours and lightexposure at 365 nm for 20 min after incubation with 0-5 μM ofsulforaphane for 24 hours. Table 2 shows that cell viability is afunction of the concentrations of the photo-oxidants and of thesulforaphane. For instance, the cell viability (9.4, 11.7, 15.2 and27.4% of the control) was dependent on the concentration of sulforaphane(0.0, 1.25, 2.5 and 5.0 μM respectively) when the cells were treatedwith 50 μM all-trans-retinal.

TABLE 2 Survival (%) of Retina Pigment Epithelium Cell Exposed toAll-trans-Retinal in light and dark and pretreated with SulforaphaneSulforaphane All-trans-Retinal (μM) (μM) 100 50 25 12.5 0 ControlExposed to 365 nm light 0.00 3.1 9.4 41.9 83.6 100.6 100.5 1.25 3.4 11.754.4 95.4 100.0 100.7 2.50 5.2 15.2 63.8 96.5 100.6 97.4 5.00 8.2 27.472.2 103.4 100.4 101.4 In Dark 0.00 75.0 96.8 97.6 96.3 97.1 99.1 1.2574.7 98.4 97.7 96.3 96.6 96.7 2.50 75.6 99.9 98.3 101.5 99.4 99.5 5.0084.6 106.8 104.8 104.6 103.1 105.6

1. A method of treating a subject in need of treatment of an oxidativestress disorder, said method comprising administering to said subject acomposition comprising a pharmaceutically effective amount of a compoundthat elevates intracellular levels of glutathione or intracelluar levelsof at least one Phase IT detoxification enzyme in diseased tissue ofsaid subject, wherein said oxidative stress disorder is selected fromthe group consisting of Alzheimer's Disease and aging.
 2. The method ofclaim 1, wherein said compound is selected from the group consisting ofan isothiocyanate and a glucosinolate.
 3. The method of claim 2, whereinsaid isothiocyante is sulforaphane.
 4. (canceled)
 5. The method of claim1, wherein said Phase IT detoxification enzyme is selected from thegroup consisting of UDP-glucuronosyltransferases, sulfotransferases,phenol-O-methyltransferase, catechol-O-methyltransferase, histamineN-methyltransferase, nicotinamide N-methyltransferase, thiopurinemethyltransferase, thiol methyltransferase, N-acetyltransferases,O-acetyltransferases, acyl-CoA synthetases, acyl-CoA:amino acidN-acyltransferases, aminoacyl-tRNA synthetases, glutathione synthetases,gamma glutamylcysteine synthetases, glutathione S-transferases, quinonereductases, heme oxygenases, rhodaneses, glutathione reductase,glutathione peroxidase, catalase and superoxide dismutase.
 6. A methodof protecting a subject from an oxidative stress disorder, said methodcomprising administering to said subject a composition comprising apharmaceutically effective amount of a compound that elevatesintracellular levels of glutathione or intracellular levels of at leastone Phase II detoxification enzyme in diseased tissue of said subject,wherein said oxidative stress disorder is selected from the groupconsisting of Alzheimer's Disease and aging.
 7. The method of claim 6,wherein said compound is selected from the group consisting of anisothiocyanate and a glucosinolate.
 8. The method of claim 7, whereinsaid isothiocyante is sulforaphane.
 9. (canceled)
 10. The method ofclaim 6, wherein said Phase II detoxification enzyme is selected fromthe group consisting of UDP-glucuronosyltransferases, sulfotransferases,phenol-O-methyltransferase, catechol-O-methyltransferase, histamineN-methyltransferase, nicotinamide N-methyltransferase, thiopurinemethyltransferase, thiol methyltransferase, N-acetyltransferases,O-acetyltransferases, acyl-CoA synthetases, acyl-CoA:amino acidN-acyltransferases, aminoacyl-tRNA synthetases, glutathione synthetases,gamma glutamylcysteine synthetases, glutathione S-transferases, quinonereductases, heme oxygenases, rhodaneses, glutathione reductase,glutathione peroxidase, catalase and superoxide dismutase. 11.-29.(canceled)
 30. The method of claim 1, wherein the composition isadministered orally, nasally, parenterally, intrasystemically,intraperitoneally, topically, bucally, or as an oral or nasal spray. 31.The method of claim 1, wherein the composition further comprises acarrier or excipient and is formulated as a lotion, a cream, a gel or anointment for topical administration.
 32. The method of claim 31, whereinthe composition is administered to the skin.
 33. The method of claim 6,wherein the composition is administered orally, nasally, parenterally,intrasystemically, intraperitoneally, topically, bucally, or as an oralor nasal spray.
 34. The method of claim 6, wherein the compositionfurther comprises a carrier or excipient and is formulated as a lotion,a cream, a gel or an ointment for topical administration.
 35. The methodof claim 34, wherein the composition is administered to the skin.